Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering

Xie, Wenping; Lv, Xiaomei; Ye, Lidan; Zhou, Pingping; Yu, Hongwei

doi:10.1016/j.ymben.2015.04.009

Cited by 185 publications

(166 citation statements)

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“…For each ORF, the ssODNs encoded an alternate start codon (GTG), a common codon, a rare codon, and a frame-shift knockout (KO) mutation. In addition to mutations that alter gene expression, we also included a protein sequence change in the crtYB lycopene-cyclase domain known to increase lycopene production (Xie et al, 2015). Lastly, we targeted terminators at putative poly-A signal sites.…”

Section: Methods Detailsmentioning

confidence: 99%

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

Barbieri

Muir

Akhuetie-Oni

et al. 2017

Cell

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SUMMARY We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing of synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with efficiencies >40% without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >105. This method was used to diversify a heterologous β-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes.

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Section: Methods Detailsmentioning

confidence: 99%

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

Barbieri

Muir

Akhuetie-Oni

et al. 2017

Cell

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“…Saccharomyces cerevisiae has been reported as a safe (Generally Recognized as Safe, GRAS) and robust host cell to produce heterologous carotenoids, including lycopene [19], β-carotene [20] and astaxanthin [21]. Thus, in our study, crocetin was successfully synthesized in S. cerevisiae through incorporating heterologous CrtZ and CCD in an existing β-carotene producing strain SyBE_Sc0014CY06 (with β-carotene titer of 220 mg/L) (Table 1).…”

Section: Introductionmentioning

confidence: 99%

Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae

et al. 2017

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BackgroundDue to excellent performance in antitumor, antioxidation, antihypertension, antiatherosclerotic and antidepressant activities, crocetin, naturally exists in Crocus sativus L., has great potential applications in medical and food fields. Microbial production of crocetin has received increasing concern in recent years. However, only a patent from EVOVA Inc. and a report from Lou et al. have illustrated the feasibility of microbial biosynthesis of crocetin, but there was no specific titer data reported so far. Saccharomyces cerevisiae is generally regarded as food safety and productive host, and manipulation of key enzymes is critical to balance metabolic flux, consequently improve output. Therefore, to promote crocetin production in S. cerevisiae, all the key enzymes, such as CrtZ, CCD and ALD should be engineered combinatorially.ResultsBy introduction of heterologous CrtZ and CCD in existing β-carotene producing strain, crocetin biosynthesis was achieved successfully in S. cerevisiae. Compared to culturing at 30 °C, the crocetin production was improved to 223 μg/L at 20 °C. Moreover, an optimal CrtZ/CCD combination and a titer of 351 μg/L crocetin were obtained by combinatorial screening of CrtZs from nine species and four CCDs from Crocus. Then through screening of heterologous ALDs from Bixa orellana (Bix_ALD) and Synechocystis sp. PCC6803 (Syn_ALD) as well as endogenous ALD6, the crocetin titer was further enhanced by 1.8-folds after incorporating Syn_ALD. Finally a highest reported titer of 1219 μg/L at shake flask level was achieved by overexpression of CCD2 and Syn_ALD. Eventually, through fed-batch fermentation, the production of crocetin in 5-L bioreactor reached to 6278 μg/L, which is the highest crocetin titer reported in eukaryotic cell.Conclusions Saccharomyces cerevisiae was engineered to achieve crocetin production in this study. Through combinatorial manipulation of three key enzymes CrtZ, CCD and ALD in terms of screening enzymes sources and regulating protein expression level (reaction temperature and copy number), crocetin titer was stepwise improved by 129.4-fold (from 9.42 to 1219 μg/L) as compared to the starting strain. The highest crocetin titer (6278 μg/L) reported in microbes was achieved in 5-L bioreactors. This study provides a good insight into key enzyme manipulation involved in serial reactions for microbial overproduction of desired compounds with complex structure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0665-1) contains supplementary material, which is available to authorized users.

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“…[40] Another group found that crtYB from Xanthophyllomyces dendrorhous performed better than crtB from P. agglomerans and employed directed evolution on the crtYB gene to reduce its lycopene cyclization activity while increasing the specificity toward phytoene synthesis in S. cerevisiae. [41] For levopimaradiene production in E. coli, the red colored lycopene was used as a colorimetric reporter for high-throughput screening of GGPP synthase mutants while levopimaradiene synthase (LPS) was engineered by rational mutagenesis (Figure 4). [42] Through the established LPS model, 15 different residues within the binding pocket were identified and single mutations were designed based on phylogeny.…”

Section: Enzyme Engineeringmentioning

confidence: 99%

“…[58] In addition, crtE gene or HMG1 gene encoding HMGR was overexpressed, enhancing lycopene production. [41,58,59] In other study, since the cytosol FPP pool partly flows into mitochondria, amorpha-4,11-diene synthase (ADS) was fused with mitochondrion-targeting signal peptide (mtADS) to utilize mitochondrial FPP as well ( Figure 6). [60] By coexpressing mtADS with the native ADS, amorphadiene production was greatly increased.…”

Section: Increasing Upstream Fluxmentioning

confidence: 99%

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

Park

Yang

et al. 2017

Adv. Biosys.

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Natural products have been attracting much interest around the world for their diverse applications, especially in drug and food industries. Plants have been a major source of many different natural products. However, plants are affected by weather and environmental conditions and their successful extraction is rather limited. Chemical synthesis is inefficient due to the complexity of their chemical structures involving enantioselectivity and regioselectivity. For these reasons, an alternative means of overproducing valuable natural products using microorganisms has emerged. In recent years, various metabolic engineering strategies have been developed for the production of natural products by microorganisms. Here, the strategies taken to produce natural products are reviewed. For convenience, natural products are classified into four main categories: terpenoids, phenylpropanoids, polyketides, and alkaloids. For each product category, the strategies for establishing and rewiring the metabolic network for heterologous natural product biosynthesis, systems approaches undertaken to optimize production hosts, and the strategies for fermentation optimization are reviewed. Taken together, metabolic engineering has enabled microorganisms to serve as a prominent platform for natural compounds production. This article examines both the conventional and novel strategies of metabolic engineering, providing general strategies for complex natural compound production through the development of robust microbial‐cell factories.

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Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering

Cited by 185 publications

References 44 publications

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes

Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

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